The current inability to perform efficient, site-specific integration of incoming DNA into the chromosomes of higher organisms is holding up advances in basic and applied biology. Recently strategies for chromosomal integration that take advantage of the high efficiency and tight sequence specificity of recombinase enzymes isolated from microorganisms have been described. In particular, a class of phage integrases that includes the φC31 integrase (Kuhstoss, S., and Rao, R. N., J. Mol. Biol. 222, 897-908 (1991); Rausch, H., and Lehmann, M., Nucleic Acids Research 19, 5187-5189 (1991)) have been shown to function in mammalian cells (Groth, A. C., et al., Proc. Natl. Acad. Sci. USA 97, 5995-6000 (2000)).
Such site-specific recombinase enzymes have long DNA recognition sites that are typically not present even in the large genomes of mammalian cells. However, it has been recently demonstrated that recombinase pseudo sites, i.e. sites with a significant degree of identity to the wild-type binding site for the recombinase, are present in these genomes (Thyagarajan, B., et al., Gene 244, 47-54 (2000)).
The present disclosure teaches methods to alter the specificity of recombinases to provide altered recombinases that can be used more effectively in genetic engineering of the chromosomes of higher cells.